Methods and materials for treating inflammatory conditions

ABSTRACT

The invention provides methods and materials for treating inflammatory conditions. Specifically, the invention provides polypeptides, isolated nucleic acids, host cells, and methods that can be used to induce an antibody response in a mammal against an antigen such as C5 or C5a. For example, the methods and materials described herein can be used to reduce the effects of C5a within a mammal by reducing the amount of total and receptor bound C5a in the mammal.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. Provisional Application Ser.No. 60/430,278, filed Dec. 2, 2002.

TECHNICAL FIELD

The invention relates to methods and materials involved in treatinginflammatory conditions.

BACKGROUND

Rheumatoid arthritis (RA) is an autoimmune, inflammatory condition thataffects peripheral joints. Collagen-induced arthritis animal models havehelped define genes related to RA conditions. A major histocompatibilitycomplex (MHC) class II gene (Aq in mouse) important for the initiationand maintenance of RA conditions has been identified. This genefunctionally corresponds to the HLA-DR gene DR*0401 in humans,suggesting that T cell mediated autoimmune recognition of joint specificantigens is involved in the disease.

Genes in regions outside the MHC also have been found to be importantfor the initiation and maintenance of RA conditions. One of these generegions is located on chromosome 2 in mouse and contains a gene codingfor the complement factor C5. One of the active components of C5 is C5a,which is released during complement binding to immunocomplexes. Therelease of C5a triggers several different pathways that lead torheumatoid inflammation. C5a produced locally in an inflammatory jointcan bind to receptors on macrophages and neutrophilic granulocytes,leading to infiltration of inflammatory cells into joints. C5 also playsa central role in complement-mediated processes such as sepsis,myocardial ischemia/reperfusion injury, adult respiratory distresssyndrome, nephritis, and graft rejection, as well as complement-mediatedinflammatory conditions such as rheumatoid arthritis, asthma,inflammatory bowel disease, multiple sclerosis, arteriosclerosis, andvasculitis.

SUMMARY

The invention relates to methods and materials for treating inflammatoryconditions such as sepsis, myocardial ischemia/reperfusion injury, adultrespiratory distress syndrome, nephritis, graft rejection, rheumatoidarthritis, asthma, inflammatory bowel disease, multiple sclerosis,arteriosclerosis, and vasculitis in a mammal. Specifically, theinvention provides polypeptides, isolated nucleic acids, host cells, andmethods for treating inflammatory conditions. Polypeptides of theinvention can be immunogenic. In one aspect, the invention providesimmunogenic polypeptides that contain both self and non-self amino acidsegments. For example, the invention provides a polypeptide thatcontains a self C5a amino acid segment and one or more non-self T cellepitopes. In another aspect, the invention provides isolated nucleicacids that encode immunogenic polypeptides suitable for treating amammal with an inflammatory condition. In addition, the inventionprovides host cells containing isolated nucleic acids encodingpolypeptides provided herein. Such host cells can be used to producelarge amounts of the encoded polypeptides, for example.

In one aspect, the invention features a composition containing apolypeptide, wherein the polypeptide includes a self C5 amino acidsegment and a non-self amino acid segment, and wherein the length of thenon-self segment is less than 350 amino acids (e.g., less than 300 aminoacids, less than 250 amino acids, or less than 200 amino acids).Administration of the polypeptide to a mammal can induces an anti-C5response in the mammal, and the genome of the mammal can include anucleic acid that encodes the self C5 amino acid segment. The non-selfamino acid segment can be a bacterial amino acid segment (e.g., an MBPamino acid segment). The non-self amino acid segment can be a C5 aminoacid segment. The non-self C5 amino acid segment can be non-naturallyoccurring. The non-self C5 amino acid segment can contain at least two Tcell epitopes. The non-self amino acid segment can be a vertebrate(e.g., mammalian) C5 amino acid segment. The non-self vertebrate C5amino acid segment can contain at least two T cell epitopes. Thenon-self amino acid segment can be a viral amino acid segment or afungal amino acid segment.

The invention also features a method for treating a mammal (e.g., ahuman) having an inflammatory condition. The method can includeadministering a polypeptide to the mammal such that the polypeptideinduces an anti-C5 response in the mammal, the polypeptide containing aself C5 amino acid segment and a non-self amino acid segment, where thegenome of the mammal contains a nucleic acid that encodes the self C5amino acid segment. The inflammatory condition can be sepsis, myocardialischemia/reperfusion injury, adult respiratory distress syndrome,nephritis, graft rejection, rheumatoid arthritis, asthma, inflammatorybowel disease, multiple sclerosis, arteriosclerosis, and/or vasculitis.The polypeptide can be MBP-C5a. The self C5 amino acid segment cancontain a portion of a C5a sequence. The non-self amino acid segment cancontain a portion of a C5 sequence. The non-self amino acid segment canbe viral, bacterial, fungal, mammalian, and/or non-naturally occurring.The non-self amino acid segment can contain at least two T cellepitopes.

In another aspect, the invention features a composition containing apolypeptide having a self C5 amino acid segment and a non-self C5 aminoacid segment. Administration of the polypeptide to a mammal can inducean anti-C5 response in the mammal, and the genome of the mammal cancontain a nucleic acid that encodes the self C5 amino acid segment. Thenon-self C5 amino acid segment can be non-naturally occurring. Thenon-self C5 amino acid segment can contain at least two T cell epitopes.

In another aspect, the invention features a composition containing apolypeptide having a self C5 amino acid segment and a non-selfvertebrate amino acid segment. Administration of the polypeptide to amammal can induce an anti-C5 response in the mammal, and the genome ofthe mammal can contain a nucleic acid that encodes the self C5 aminoacid segment. The non-self vertebrate amino acid segment can be amammalian amino acid segment. The non-self vertebrate amino acid segmentcan contain at least two T cell epitopes.

In another aspect, the invention features a composition containing apolypeptide that includes a self C5 amino acid segment and a non-selfamino acid segment, wherein the length of the non-self amino acidsegment is less than 350 (e.g., less than 300, less than 250, or lessthan 200) amino acid residues. Administration of the polypeptide to amammal can induce an anti-C5 response in the mammal, and the genome ofthe mammal can contain a nucleic acid that encodes the self C5 aminoacid segment. The non-self amino acid segment can be viral, bacterial,fungal, mammalian, and/or non-naturally occurring. The non-selfmammalian amino acid segment can contain at least two T cell epitopes.

In another aspect, the invention features a composition containing apolypeptide and an adjuvant, wherein the polypeptide contains a self C5amino acid segment and a bacterial amino acid segment. The bacterialamino acid segment can be MBP.

In another aspect, the invention features a composition containing apolypeptide having a self C5 amino acid segment and a fungal amino acidsegment.

In another aspect, the invention features a composition containing apolypeptide having a self C5 amino acid segment and a viral amino acidsegment.

In another aspect, the invention features an isolated nucleic acidencoding a polypeptide that contains a self C5 amino acid segment and anon-self C5 amino acid segment.

In another aspect, the invention features an isolated nucleic acidencoding a polypeptide that contains a self C5 amino acid segment and anon-self vertebrate amino acid segment.

In yet another aspect, the invention features an isolated nucleic acidencoding a polypeptide that contains a self C5 amino acid segment and anon-self amino acid segment, the length of the non-self amino acidsegment being less than 360 (e.g., less than 300, less than 250, or lessthan 200) amino acid residues.

In another aspect, the invention features an isolated nucleic acidencoding a polypeptide that contains a self C5 amino acid segment and abacterial amino acid segment, wherein the polypeptide lacks a factor Xacleavage site between the C5 amino acid segment and the bacterial aminoacid segment. The non-self bacterial amino acid segment can be MBP.

In another aspect, the invention features an isolated nucleic acidencoding a polypeptide that contains a self C5 amino acid segment and afungal amino acid segment.

In another aspect, the invention features an isolated nucleic acidencoding a polypeptide that contains a self C5 amino acid segment and aviral amino acid segment.

Another aspect of the invention features a host cell containing (1) anisolated nucleic acid, where the isolated nucleic acid encodes apolypeptide having a self C5 amino acid segment and a non-self C5 aminoacid segment; (2) an isolated nucleic acid, where the isolated nucleicacid encodes a polypeptide having a self C5 amino acid segment and anon-self vertebrate amino acid segment; (3) an isolated nucleic acid,where the isolated nucleic acid encodes a polypeptide having a self C5amino acid segment and a non-self amino acid segment, the length of thenon-self amino acid segment being less than 350 (e.g., less than 300,less than 250, or less than 200) amino acid residues; (4) an isolatednucleic acid, where the isolated nucleic acid encodes a polypeptidehaving a self C5 amino acid segment and a non-self bacterial amino acidsegment; (5) an isolated nucleic acid, where the isolated nucleic acidencodes a polypeptide having a self C5 amino acid segment and a non-selffungal amino acid segment; and/or (6) an isolated nucleic acid, wherethe isolated nucleic acid encodes a polypeptide having a self C5 aminoacid segment and a non-self viral amino acid segment. The host cell canexpress the polypeptide.

In another aspect, the invention features a composition foradministration to a mammal. The composition can contain a polypeptide,wherein with respect to the mammal the polypeptide contains a self C5amino acid segment and a non-self C5 amino acid segment. Administrationof the polypeptide to a mammal can induce an anti-C5 response in themammal, wherein the genome of the mammal contains a nucleic acid thatencodes the self C5 amino acid segment. The non-self C5 amino acidsegment can be non-naturally occurring. The non-self C5 amino acidsegment can contain at least two T cell epitopes.

In another aspect, the invention features a composition foradministration to a mammal, the composition containing a polypeptide,wherein with respect to the mammal the polypeptide includes a self C5amino acid segment and a non-self vertebrate amino acid segment.Administration of the polypeptide to a mammal can induce an anti-C5response in the mammal, wherein the genome of the mammal contains anucleic acid that encodes the self C5 amino acid segment. The non-selfvertebrate amino acid segment can be a mammalian amino acid segment. Thenon-self vertebrate amino acid segment can include at least two T cellepitopes.

In yet another aspect, the invention features a composition foradministration to a mammal. The composition can contain a polypeptide,wherein with respect to the mammal the polypeptide includes a self C5amino acid segment and a non-self amino acid segment, wherein the lengthof the non-self amino acid segment is less than 350 (e.g., less than300, less than 250, or less than 200) amino acid residues.Administration of the polypeptide to a mammal can induce an anti-C5response in the mammal, wherein the genome of the mammal contains anucleic acid that encodes the self C5 amino acid segment. The non-selfamino acid segment can be viral, bacterial, fungal, or mammalian. Thenon-self amino acid segment can be non-naturally occurring. The non-selfmammalian amino acid segment can include at least two T cell epitopes.

In another aspect, the invention features a composition foradministration to a mammal, the composition containing a polypeptide andan adjuvant, wherein with respect to the mammal the polypeptide includesa self C5 amino acid segment and a bacterial amino acid segment (e.g.,MBP).

In another aspect, the invention features a composition foradministration to a mammal, the composition containing a polypeptide,wherein with respect to the mammal the polypeptide includes a self C5amino acid segment and a fungal amino acid segment.

The invention also features a composition for administration to amammal, the composition containing a polypeptide, wherein with respectto the mammal the polypeptide includes a self C5 amino acid segment anda viral amino acid segment.

In another aspect, the invention features an isolated nucleic acid,wherein the isolated nucleic acid encodes a polypeptide foradministration to a mammal, and wherein with respect to the mammal thepolypeptide includes a self C5 amino acid segment and a non-self C5amino acid segment.

The invention also features an isolated nucleic acid, wherein theisolated nucleic acid encodes a polypeptide for administration to amammal, and wherein with respect to the mammal the polypeptide containsa self C5 amino acid segment and a non-self vertebrate amino acidsegment.

In another aspect, the invention features an isolated nucleic acid,wherein the isolated nucleic acid encodes a polypeptide foradministration to a mammal, wherein with respect to the mammal thepolypeptide includes a self C5 amino acid segment and a non-self aminoacid segment, and wherein the length of the non-self amino acid segmentis less than 350 (e.g., less than 300, less than 250, or less than 200)amino acid residues.

In still another aspect, the invention features an isolated nucleicacid, wherein the isolated nucleic acid encodes a polypeptide foradministration to a mammal, wherein with respect to the mammal thepolypeptide contains a self C5 amino acid segment and a bacterial aminoacid segment, and wherein the polypeptide lacks a factor Xa cleavagesite between the C5 amino acid segment and the bacterial amino acidsegment. The bacterial amino acid segment can be MBP.

In another aspect, the invention features an isolated nucleic acid,wherein the isolated nucleic acid encodes a polypeptide foradministration to a mammal, and wherein with respect to the mammal thepolypeptide includes a self C5 amino acid segment and a fungal aminoacid segment.

The invention also features an isolated nucleic acid, wherein theisolated nucleic acid encodes a polypeptide for administration to amammal, and wherein with respect to the mammal the polypeptide containsa self C5 amino acid segment and a viral amino acid segment.

The invention also features a host cell containing (1) an isolatednucleic acid, where the isolated nucleic acid encodes a polypeptide foradministration to a mammal, wherein with respect to the mammal thepolypeptide includes a C5 amino acid segment and a non-self C5 aminoacid segment; (2) an isolated nucleic acid, where the isolated nucleicacid encodes a polypeptide for administration to a mammal, wherein withrespect to the mammal the polypeptide includes a self C5 amino acidsegment and a non-self vertebrate amino acid C5 segment; (3) an isolatednucleic acid, where the isolated nucleic acid encodes a polypeptide foradministration to a mammal, wherein with respect to the mammal thepolypeptide includes a self C5 amino acid segment and a non-self aminoacid segment, the length of the non-self amino acid segment being lessthan 350 (e.g., less than 300, less than 250, or less than 200) aminoacid residues; (4) an isolated nucleic acid, where the isolated nucleicacid encodes a polypeptide for administration to a mammal, wherein withrespect to the mammal the polypeptide includes a self C5 amino acidsegment and a non-self bacterial amino acid segment; (5) an isolatednucleic acid, where the isolated nucleic acid encodes a polypeptide foradministration to a mammal, wherein with respect to the mammal thepolypeptide includes a self C5 amino acid segment and a non-self fungalamino acid segment; and/or (6) an isolated nucleic acid, where theisolated nucleic acid encodes a polypeptide for administration to amammal, wherein with respect to the mammal the polypeptide includes aself C5 amino acid segment and a non-self viral amino acid segment. Thehost cell can express the polypeptide.

In yet another aspect, the invention features a composition foradministration to a mammal. The composition can contain a polypeptide,wherein with respect to the mammal the polypeptide contains a self C5amino acid segment and a non-self amino acid segment, and wherein theself C5 amino acid segment is at least 90 percent identical to a C5sequence from the mammal.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing drawings and detailed description, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a depiction of a mouse pro-C5 DNA sequence (SEQ ID NO:1).

FIG. 2 is a depiction of the amino acid sequence of mouse pro-C5including the signal peptide (SEQ ID NO:2).

FIG. 3 is a diagram depicting a nucleic acid vector designed to expressa fusion polypeptide containing maltose binding protein (MBP) and mouseC5a.

FIG. 4 is the nucleic acid sequence of a MBP-C5a PCR product (SEQ IDNO:3).

FIG. 5 is the amino acid sequence of a MBP-C5a fusion polypeptide (SEQID NO:4).

FIG. 6 is a graph plotting the incidence of collagen-induced arthritisin control mice (open diamonds) and in mice vaccinated with a MBP-C5afusion polypeptide (filled circles). *, p<0.05; **, p<0.01; ***,p<0.005.

FIG. 7 is a graph plotting the mean arthritis score for collagen-inducedarthritis in control mice (open circles) and in mice vaccinated with aMBP-C5a fusion polypeptide (filled circles). *, p<0.05; **, p<0.01; ***,p<0.005.

FIG. 8 is a graph plotting the mean maximum arthritis score forcollagen-induced arthritis in control mice and in mice vaccinated with aMBP-C5a fusion polypeptide. **, p<0.01.

FIG. 9 is a graph plotting the area under the curve for arthritis scoredata obtained from mice injected with collagen and then treated with PBC(control) or a MBP-C5a fusion polypeptide. ***, p<0.005.

FIG. 10 is a graph plotting the percent incidence of chroniccollagen-induced arthritis in control mice (filled circles) and in micevaccinated with a MBP-C5a fusion polypeptide (open diamonds). *, p<0.05;**, p<0.01.

FIG. 11 is a graph plotting the mean score for chronic collagen-inducedarthritis in control mice (filled circles) and in mice vaccinated with aMBP-C5a fusion polypeptide (open circles). *, p<0.05.

DETAILED DESCRIPTION

The invention provides methods and materials for treating inflammatoryconditions. The term “inflammatory condition” as used herein refers to adisease, disease state, syndrome, or other condition resulting ininflammation. For example, rheumatoid arthritis and asthma areinflammatory conditions. Other examples of inflammatory conditionsinclude, without limitation, sepsis, myocardial ischemia/reperfusioninjury, adult respiratory distress syndrome, nephritis, graft rejection,inflammatory bowel disease, multiple sclerosis, arteriosclerosis, andvasculitis. The invention provides polypeptides, isolated nucleic acids,host cells, and methods for inducing an antibody response in a mammalagainst an antigen such as C5 or C5a. For example, the methods andmaterials described herein can be used to reduce the effects of C5a in amammal by reducing the amount of total and/or receptor bound C5a.

Polypeptides

The invention provides polypeptides that can be used to treat aninflammatory condition. Such polypeptides can be immunogenic. An“immunogenic” polypeptide is any polypeptide that effectively induces anantibody response in a mammal. For example, an immunogenic polypeptidecan be a polypeptide that effectively induces the production of ananti-self C5 antibody in a mammal.

Polypeptides of the invention can contain at least one amino acidsegment that would be considered non-self to the particular mammalreceiving the polypeptide. For example, a polypeptide that inducesproduction of an anti-self C5 antibody can contain a self C5 amino acidsegment and a non-self amino acid segment (e.g., a non-self C5 aminoacid segment). The self C5 amino acid segment can confer the specificityof the anti-self C5 antibody response, and the non-self amino acidsegment can enhance the immunogenicity of the polypeptide. The non-selfamino acid segment (e.g., a non-self C5 amino acid segment) can containat least two T cell epitopes. Alternatively, the non-self amino acidsegment can stabilize an immunogenic polypeptide such that the specificanti-self C5 antibody response is induced. The self C5 amino acidsegment of a polypeptide can be a portion of C5 that directly interactswith a C5a receptor. Examples of such self C5 amino acid segmentsinclude, without limitation, C5a or fragments of C5a. Additionally, theself C5 amino acid segment of a polypeptide can be a portion of C5 thatindirectly influences the interaction of C5a with a C5a receptor. Forexample, a polypeptide containing the C5 convertase recognition sequenceof C5 can induce the production of antibodies that bind to the C5convertase recognition sequence of C5, thereby inhibiting the conversionof C5 to C5a by C5 convertase.

The term “amino acid segment” as used herein refers to a contiguousstretch of amino acids within a polypeptide. For example, the amino acidresidues 30 to 40 within a 100 amino acid polypeptide would beconsidered an amino acid segment. An amino acid segment can be anylength greater than eight amino acid residues (e.g., greater than aboutnine, ten, 15, 20, 25, 30, 40, 50, 75, 100, 150, 200, 500, 1000, or moreamino acid residues). Thus, an amino acid segment can be C5, the entireC5a region of C5, or a portion of C5a. In some embodiments, an aminoacid segment can have a length less than 1000 amino acid residues (e.g.,less than 500, less than 400, less than 350, less than 300, less than200, or less than 100 amino acid residues). In other embodiments, anamino acid segment can have a length from about 20 to about 200 aminoacid residues (e.g., about 30 to about 180 amino acid residues, or about40 to about 150 amino acid residues).

The term “self” as used herein with respect to an amino acid segment anda particular mammal generally refers to any amino acid segment that theparticular mammal possesses endogenously. If an amino acid segment isderived from a member of one species and introduced into another memberof the same species that endogenously possesses the amino acid segment,then that particular amino acid segment is considered a self amino acidsegment. For example, a C5 amino acid segment derived from a mouse is aself C5 amino acid segment when introduced into that same mouse, agenetically identical mouse, or a non-genetically identical mouse thatendogenously possesses the same amino acid segment.

The term “non-self” as used herein with respect to an amino acid segmentand a particular mammal generally refers to any amino acid segment thatthe particular mammal does not possess endogenously. If an amino acidsegment is derived from a member of a first species and introduced intoa member of a second species that does not endogenously possess theamino acid segment, then that particular amino acid segment can beconsidered a non-self amino acid segment. For example, a C5 amino acidsegment derived from a mouse can be considered a non-self C5 amino acidsegment when introduced into a human. In another example, if apolypeptide contains a C5a amino acid segment from a mouse and a C5bamino acid segment from a human, and that polypeptide is introduced intoa mouse, then the C5a amino acid segment can be considered a self C5aamino acid segment and the C5b amino acid segment can be considered anon-self C5b amino acid segment. Alternatively, if the same polypeptideis introduced into a human, the C5a amino acid segment can be considereda non-self C5a amino acid segment and the C5b amino acid segment can beconsidered a self C5b amino acid segment. If an amino acid segment fromone member of a species is considered polymorphic to another member ofthe same species, then that amino acid segment can be considered anon-self amino acid segment to a member of the species not possessingthat polymorphism. For example, if a C5 amino acid segment from a humanhaving one type of polymorphism in the amino acid segment is introducedinto a second human that does not have that particular type ofpolymorphism, the C5 amino acid segment can be considered a non-selfamino acid segment to the second human. It also will be understood thatcryptic T cell epitopes (i.e., self peptides that under normalconditions are not expressed on MHC molecules to the level required forrecognition by T cells) can be considered non-self.

The non-self and self amino acid segments can be from either the same ordifferent naturally-occurring polypeptides. For example, if apolypeptide contains a self amino acid segment from human C5, then thenon-self amino acid segment can be from either the same type ofpolypeptide (e.g., rat C5) or a different type of polypeptide (e.g., ratalbumin). In another example, a polypeptide can contain a self C5a aminoacid segment and one or more non-self T cell epitopes provided by MBP.Further, an amino acid segment can be from any type of polypeptideincluding, without limitation, a bacterial polypeptide (e.g., MBP), afungal polypeptide, a viral polypeptide, or a mammalian polypeptide.

The self segment or segments of the polypeptides provided hereintypically are at least 90 percent identical (e.g., at least 91, 92, 93,94, 95, 96, 97, 98, or 99 percent identical) to a sequence from apolypeptide found in the mammal to which the polypeptide will beadministered. In some embodiments, the self segment can be 100 percentidentical to a sequence from a polypeptide found in the mammal to whichthe polypeptide will be administered. The invention thus providespolypeptides that contain an amino acid segment having (1) a length, and(2) a percent identity to a reference amino acid sequence (e.g., anamino acid sequence from a particular mammal) over that length. Theinvention also provides isolated nucleic acid molecules that contain anucleic acid sequence encoding a polypeptide that contains an amino acidsegment having (1) a length, and (2) a percent identity to a mammal'samino acid sequence over that length. Typically, the mammalian aminoacid or nucleic acid sequence is a referred to as a reference sequence,and the amino acid or nucleic acid sequence being compared to themammalian sequence is referred to as the target sequence. For example, amammal's sequence can be the reference sequence having the sequence setforth in SEQ ID NO:2.

A length and percent identity over that length for any amino acid ornucleic acid sequence is determined as follows. First, an amino acid ornucleic acid sequence is compared to an amino acid or nucleic acidsequence from the mammal to which it will be administered using theBLAST 2 Sequences (Bl2seq) program from the stand-alone version ofBLASTZ containing BLASTN version 2.0.14 and BLASTP version 2.0.14. Thisstand-alone version of BLASTZ can be obtained from Fish & Richardson'sweb site (World Wide Web at “fr” dot “com” slash “blast”), the U.S.government's National Center for Biotechnology Information web site(World Wide Web at “ncbi” dot “nlm” dot “nih” dot “gov”), or the StateUniversity of New York at Old Westbury Library (QH 497.m6714).Instructions explaining how to use the Bl2seq program can be found inthe readme file accompanying BLASTZ.

Bl2seq performs a comparison between two sequences using either theBLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acidsequences, while BLASTP is used to compare amino acid sequences. Tocompare two nucleic acid sequences, the options are set as follows: —iis set to a file containing the first nucleic acid sequence to becompared (e.g., C:\seq1.txt); —j is set to a file containing the secondnucleic acid sequence to be compared (e.g., C:\seq2.txt); —p is set toblastn; —o is set to any desired file name (e.g., C:\output.txt); —q isset to −1; —r is set to 2; and all other options are left at theirdefault settings. For example, the following command can be used togenerate an output file containing a comparison between two sequences:C:\Bl2seq —i c:\seq1.txt —j c:\seq2.txt —p blastn —o c:\output.txt —q −1—r 2. To compare two amino acid sequences, the options of Bl2seq are setas follows: —i is set to a file containing the first amino acid sequenceto be compared (e.g., C:\seq1.txt); —j is set to a file containing thesecond amino acid sequence to be compared (e.g., C:\seq2.txt); —p is setto blastp; —o is set to any desired file name (e.g., C:\output.txt); andall other options are left at their default settings. For example, thefollowing command can be used to generate an output file containing acomparison between two amino acid sequences: C:\Bl2seq —i c:\seq1.txt —jc:\seq2.txt —p blastp —o c:\output.txt. If the target sequence shareshomology with any portion of the mammalian sequence, then the designatedoutput file will present those regions of homology as aligned sequences.If the target sequence does not share homology with any portion of themammalian sequence, then the designated output file will not presentaligned sequences.

Once aligned, a length is determined by counting the number ofconsecutive nucleotides or amino acid residues from the target sequencepresented in alignment with sequence from the mammalian sequence. Amatched position is any position where an identical nucleotide or aminoacid residue is presented in both the target and mammalian sequence.Gaps presented in the target sequence are not counted since gaps are notnucleotides or amino acid residues. Likewise, gaps presented in themammalian sequence are not counted since target sequence nucleotides oramino acid residues are counted, not nucleotides or amino acid residuesfrom the mammalian sequence.

The percent identity over a determined length is determined by countingthe number of matched positions over that length and dividing thatnumber by the length followed by multiplying the resulting value by 100.For example, if (1) a 300 amino acid target sequence is compared to areference sequence, (2) the Bl2seq program presents 200 consecutiveamino acids from the target sequence aligned with a region of thereference sequence, and (3) the number of matches over those 200 alignedamino acids is 180, then that 300 amino acid target sequence contains anamino acid segment that has a length of 200 and a percent identity overthat length of 90 (i.e., 180÷200*100=90).

It is noted that the percent sequence identity value is rounded to thenearest tenth. For example, 75.11, 75.12, 75.13, and 75.14 is roundeddown to 75.1, while 75.15, 75.16, 75.17, 75.18, and 75.19 is rounded upto 75.2. It is also noted that the length value will always be aninteger.

The non-self segment or segments of the polypeptides provided hereintypically are less than 95 percent identical (e.g., less than 94, 93,92, 91, 90, 85, 80, 75, 70, 65, 60, 55, or 50 percent identical) to asequence from a polypeptide found in the mammal to which the polypeptidewill be administered.

Any method can be used to make a polypeptide including, for example,expression by prokaryotic systems, expression by eukaryotic systems, andchemical synthesis techniques. Any method can be used to purify apolypeptide including, without limitation, fractionation,centrifugation, and chromatography. For example, polypeptides containingmaltose binding protein (MBP) can be purified using amylose affinitychromatography.

Nucleic Acids

The invention provides isolated nucleic acids encoding polypeptides suchas those described herein (e.g., polypeptides containing self andnon-self amino acid segments). For example, a nucleic acid of theinvention can encode a polypeptide having the amino acid sequence setforth in SEQ ID NO:2. Alternatively, a nucleic acid of the invention canencode a polypeptide having an amino acid sequence that contains aportion of the sequence set forth in SEQ ID NO:2. In another embodiment,a nucleic acid provided herein can encode a polypeptide having the aminoacid sequence set forth in SEQ ID NO:4. An isolated nucleic acid alsocan encode a polypeptide containing a self C5 amino acid segment and anon-self amino acid segment (e.g., a non-self C5 amino acid segment, ora non-self vertebrate, bacterial, fungal, or viral amino acid segment).The self segment encoded by the isolated nucleic acid can contain anamino acid segment (e.g., a C5 amino acid segment) with at least 90percent identity (e.g., at least 91, 92, 93, 94, 95, 96, 97, 98, or 99percent identity, or 100 percent identity) to an amino acid sequencefrom a polypeptide found in the mammal to which the polypeptide will beadministered. The non-self segment encoded by the isolated nucleic acidcan contain an amino acid segment with less than 95 percent identity(e.g., less than 94, 93, 92, 91, 90, 85, 80, 75, 70, 65, 60, 55, or 50percent identity) to an amino acid sequence from a polypeptide found inthe mammal to which the polypeptide will be administered.

The term “nucleic acid” as used herein encompasses both RNA and DNA,including cDNA, genomic DNA, and synthetic (e.g., chemicallysynthesized) DNA. The nucleic acid can be double-stranded orsingle-stranded. Where single-stranded, the nucleic acid can be thesense strand or the antisense strand. In addition, nucleic acid can becircular or linear.

The term “isolated” as used herein with reference to a nucleic acidrefers to a naturally-occurring nucleic acid that is not immediatelycontiguous with one or both of the sequences with which it isimmediately contiguous (one at the 5′ end and one at the 3′ end) in thenaturally-occurring genome of the organism from which it is derived. Forexample, an isolated nucleic acid can be, without limitation, arecombinant DNA of any length, provided one of the nucleic acidsequences normally found immediately flanking that recombinant DNA in anaturally-occurring genome is removed or absent. Thus, an isolatednucleic acid includes, without limitation, a recombinant DNA that isindependent of other sequences (e.g., a cDNA or a genomic DNA fragmentproduced by PCR or restriction endonuclease treatment), as well asrecombinant DNA that is incorporated into a vector, an autonomouslyreplicating plasmid, a virus (e.g., a retrovirus, adenovirus, or herpesvirus), or into the genomic DNA of a prokaryote or eukaryote. Inaddition, an isolated nucleic acid can include a recombinant DNA that ispart of a hybrid or fusion nucleic acid sequence.

The term “isolated” as used herein with reference to a nucleic acid alsoincludes any non-naturally-occurring nucleic acid sincenon-naturally-occurring nucleic acid sequences are not found in natureand do not have immediately contiguous sequences in a naturallyoccurring genome. For example, a non-naturally-occurring nucleic acidsuch as an engineered nucleic acid is considered to be an isolatednucleic acid. Engineered nucleic acid can be made using common molecularcloning or chemical nucleic acid synthesis techniques. Isolatednon-naturally-occurring nucleic acid can be independent of othersequences, or incorporated into a vector, an autonomously replicatingplasmid, a virus (e.g., a retrovirus, adenovirus, or herpes virus), orthe genomic DNA of a prokaryote or eukaryote. In addition, anon-naturally-occurring nucleic acid can include a nucleic acid that ispart of a hybrid or fusion nucleic acid sequence.

A nucleic acid existing among hundreds to millions of other nucleicacids within, for example, cDNA or genomic libraries, or gel slicescontaining a genomic DNA restriction digest is not to be considered anisolated nucleic acid.

The term “exogenous” as used herein with reference to a nucleic acid anda particular cell refers to any nucleic acid that does not originatefrom that particular cell as found in nature. Thus, anynon-naturally-occurring nucleic acid is considered to be exogenous to acell once introduced into the cell. It is important to note that anon-naturally-occurring nucleic acid can contain nucleic acid sequencesor fragments of nucleic acid sequences that are found in nature,provided the nucleic acid as a whole does not exist in nature. Forexample, a nucleic acid containing a genomic DNA sequence within anexpression vector is a non-naturally-occurring nucleic acid, and thus isexogenous to a cell once introduced into the cell since that nucleicacid as a whole (genomic DNA plus vector DNA) does not exist in nature.Thus, any vector, autonomously replicating plasmid, or virus (e.g.,retrovirus, adenovirus, or herpes virus) that as a whole does not existin nature is considered to be a non-naturally-occurring nucleic acid. Itfollows that genomic DNA fragments produced by PCR or restrictionendonuclease treatment as well as cDNAs are considered to benon-naturally-occurring nucleic acids since they exist as separatemolecules not found in nature. It also follows that any nucleic acidcontaining a promoter sequence and polypeptide-encoding sequence (e.g.,cDNA or genomic DNA) in an arrangement not found in nature is anon-naturally-occurring nucleic acid.

A nucleic acid that is naturally occurring can be exogenous to aparticular cell. For example, an entire chromosome isolated from a cellof person X is an exogenous nucleic acid with respect to a cell ofperson Y once that chromosome is introduced into Y's cell.

Isolated nucleic acids can be obtained using any method including,without limitation, common molecular cloning and chemical nucleic acidsynthesis techniques. For example, PCR can be used to obtain an isolatednucleic acid containing a nucleic acid sequence having similarity to thesequence set forth in SEQ ID NO:1. PCR refers to a procedure ortechnique in which target nucleic acid is amplified in a manner similarto that described in U.S. Pat. No. 4,683,195, and subsequentmodifications of the procedure described therein. Generally, sequenceinformation from the ends of the region of interest or beyond are usedto design oligonucleotide primers that are identical or similar insequence to opposite strands of a potential template to be amplified.Using PCR, a nucleic acid sequence can be amplified from RNA or DNA. Forexample, a nucleic acid sequence can be isolated by PCR amplificationfrom total cellular RNA, total genomic DNA, or cDNA, as well as frombacteriophage sequences, plasmid sequences, viral sequences, and thelike. When using RNA as a source of template, reverse transcriptase canbe used to synthesize complimentary DNA strands.

Isolated nucleic acids also can be obtained by mutagenesis. For example,an isolated nucleic acid containing a sequence set forth in SEQ ID NO:1can be mutated using common molecular cloning techniques (e.g.,site-directed mutagenesis). Possible mutations include, withoutlimitation, deletions, insertions, and substitutions, as well ascombinations of deletions, insertions, and substitutions.

In addition, nucleic acid and amino acid databases (e.g., GenBank®) canbe used to obtain an isolated nucleic acid. For example, any nucleicacid sequence having some homology to a sequence set forth in SEQ IDNO:1, or any amino acid sequence having some homology to a sequence setforth in SEQ ID NO:2 can be used as a query to search GenBank®.

Further, nucleic acid hybridization techniques can be used to obtain anisolated nucleic acid. Briefly, any nucleic acid having some homology toa sequence set forth in SEQ ID NO:1 can be used as a probe to identify asimilar nucleic acid by hybridization under conditions of moderate tohigh stringency. Once identified, the nucleic acid then can be purified,sequenced, and analyzed to determine whether it is within the scope ofthe invention as described herein.

Hybridization can be done by Southern or Northern analysis to identify aDNA or RNA sequence, respectively, which hybridizes to a probe. Theprobe can be labeled with biotin, digoxygenin, an enzyme, or aradioisotope such as ³²P The DNA or RNA to be analyzed can beelectrophoretically separated on an agarose or polyacrylamide gel,transferred to nitrocellulose, nylon, or another suitable membrane, andhybridized with the probe using standard techniques well known in theart such as those described in sections 7.39-7.52 of Sambrook et al.,(1989) Molecular Cloning, second edition, Cold Spring harbor Laboratory,Plainview, N.Y.

Further, any method can be used to direct the transcription ortranslation of a particular isolated nucleic acid encoding apolypeptide. Such methods include, without limitation, constructing anucleic acid such that a regulatory element promotes expression of anucleic acid sequence that encodes a polypeptide. Typically, regulatoryelements are DNA sequences that regulate the expression of other DNAsequences at the level of transcription. Thus, regulatory elementsinclude, without limitation, promoters, enhancers, and the like.

Host Cells

The invention provides host cells containing at least one isolatednucleic acid described herein. Such cells can be prokaryotic cells oreukaryotic cells. It is noted that cells containing an isolated nucleicacid within the scope of the invention are not required to express apolypeptide. In addition, the isolated nucleic acid can be integratedinto the genome of the cell or maintained in an episomal state. Thus,host cells can be stably or transiently transfected with a constructcontaining an isolated nucleic acid of the invention.

The host cells provided herein can contain an exogenous nucleic acidthat encodes a polypeptide. For example, cells can contain a nucleicacid encoding a self C5 amino acid segment and a non-self amino acidsegment. In addition, the host cells can express the encodedpolypeptide.

Any method can be used to introduce an isolated nucleic acid into a cellin vivo or in vitro. For example, calcium phosphate precipitation,electroporation, heat shock, lipofection, microinjection, andviral-mediated nucleic acid transfer are common methods that can be usedto introduce an isolated nucleic acid into a cell. In addition, nakedDNA can be delivered directly to cells in vivo as describe elsewhere(see, e.g., U.S. Pat. Nos. 5,580,859 and 5,589,466, and continuationsthereof). Further, isolated nucleic acids can be introduced into cellsby generating transgenic animals.

Transgenic animals can be aquatic animals (such as fish, sharks,dolphins, and the like), farm animals (such as pigs, goats, sheep, cows,horses, rabbits, and the like), rodents (such as mice, guinea pigs, andrats), non-human primates (such as baboon, monkeys, and chimpanzees),and domestic animals (such as dogs and cats). Several techniques knownin the art can be used to introduce isolated nucleic acids into animalsto produce the founder lines of transgenic animals. Such techniquesinclude, without limitation, pronuclear microinjection (U.S. Pat. No.4,873,191); retrovirus mediated gene transfer into germ lines (Van derPutten et al., Proc. Natl. Acad. Sci., USA, 82:6148 (1985)); genetransfection into embryonic stem cells (Gossler et al., Proc Natl. Acad.Sci. USA 83:9065-9069 (1986)); gene targeting into embryonic stem cells(Thompson et al., Cell, 56:313 (1989)); nuclear transfer of somaticnuclei (Schnieke et al., Science 278:2130-2133 (1997)); andelectroporation of embryos (Lo Mol. Cell. Biol., 3:1803-1814 (1983)).Once obtained, transgenic animals can be replicated using traditionalbreeding or animal cloning.

Any method can be used to identify cells containing an isolated nucleicacid of the invention. Such methods include, without limitation, PCR andnucleic acid hybridization techniques such as Northern and Southernanalysis. In some cases, immunohistochemistry and biochemical techniquescan be used to determine if a cell contains a particular isolatednucleic acid by detecting the expression of a polypeptide encoded bythat particular nucleic acid.

Methods for Treating Inflammatory Conditions

The polypeptides provided herein can be used in the manufacture of amedicament or composition for treating inflammatory conditions. Thus,the invention provides methods for treating inflammatory conditions.Such methods include, without limitation, administering a composition toa mammal. A composition can contain a polypeptide that acts as anantigen against which an immune response is desired. Further, acomposition can contain more than one polypeptide, or any combination ofdifferent polypeptides. For example, a composition can contain bothviral polypeptides and mammalian polypeptides. It is noted that eachpolypeptide in a composition can have an identical amino acid sequence.In addition, the polypeptides in a composition can contain differentamino acid segments, each of which can act as a defined antigenic unitagainst which an immune response is desired. Thus, the polypeptides in acomposition can contain different amino acid segments that correspond toany region from a polypeptide including, without limitation, receptorbinding regions, ligand binding regions, enzyme active sites, enzymecleavage sites of polypeptide substrates, antigen-binding regions ofantibodies, and epitopes recognized by antibodies. For example, thepolypeptides in a composition can encompass three different amino acidsegments, each of which corresponds to the C5 convertase recognitionsequence of C5. In addition, different or identical amino acid segmentscan be in tandem or dispersed throughout the same polypeptide.Typically, the administration of a polypeptide results in the formationof antibodies having specificity for an epitope or combination ofepitopes formed by the amino acid segments within one or more of thepolypeptides in the composition.

A composition can contain an isolated nucleic acid designed to express aparticular polypeptide when introduced into a host cell. For example, anisolated nucleic acid can be designed to encode a polypeptide having aself C5a amino acid segment and one or more than one non-self T cellepitope. Once constructed, the isolated nucleic acid can be introducedinto a host cell such that the encoded polypeptide is expressed. Anyhost cell can be used including, without limitation, prokaryotic cells(e.g., bacteria) and eukaryotic cells (e.g., human cells). Onceproduced, the polypeptide can be purified and used to vaccinate amammal. Alternatively, a composition containing an isolated nucleic aciddesigned to express a particular polypeptide can be administered to amammal such that the polypeptide is expressed in vivo.

A composition can be made by combining any of the polypeptides (e.g.,immunogenic polypeptides) or isolated nucleic acids (e.g., nucleic acidsencoding immunogenic polypeptides) provided herein with apharmaceutically acceptable carrier. Such carriers can include, withoutlimitation, sterile aqueous or non-aqueous solutions, suspensions, andemulsions. Examples of non-aqueous solvents include mineral oil,propylene glycol, polyethylene glycol, vegetable oils, and injectableorganic esters, for example. Aqueous carriers include, withoutlimitation, water, alcohol, saline, and buffered solutions.Preservatives, flavorings, and other additives such as, for example,antimicrobials, anti-oxidants, chelating agents, inert gases, and thelike also may be present. It will be appreciated that any materialdescribed herein that is to be administered to a mammal can contain oneor more pharmaceutically acceptable carriers.

A composition also can include an adjuvant. An “adjuvant” is animmunological compound that can enhance an immune response against aparticular antigen such as a polypeptide. Adjuvants such as, forexample, alum and other aluminum-based compounds (e.g., Al₂O₃) can becombined with a polypeptide containing a self amino acid segment (e.g.,a self C5 amino acid segment) to form a composition that elicits ananti-self response when administered to a mammal. Aluminum-basedcompounds can be obtained from various commercial suppliers. Forexample, REHYDRAGEL® adjuvants can be obtained from Reheis Inc.(Berkeley Heights, N.J.). REHYDRAGEL® adjuvants are based on crystallinealuminum oxyhydroxide, and are hydrated gels containing crystallineparticles with a large surface area (about 525 m²/g). Their Al₂O₃content typically ranges from about 2 percent to about 10 percent.Rehydragel LG, for example, has an Al₂O₃ content of about 6 percent, andflows readily upon slight agitation. Rehydragel LG also has a proteinbinding capacity of 1.58 (i.e., 1.58 mg of bovine serum albumin boundper 1 mg of Al₂O₃), a sodium content of 0.02 percent, a chloride contentof 0.28 percent, undetectable sulphate, an arsenic level less than 3ppm, a heavy metal content less than 15 ppm, a pH of 6.5, and aviscosity of 1090 cp. Rehydragel LG can be combined with a polypeptidesolution (e.g., a polypeptide in PBS) to yield Al(OH)₃. In addition,ALHYDROGEL™, an aluminum hydroxy gel adjuvant, (Alhydrogel 1.3%,Alhydrogel 2.0%, or Alhydrogel “85”) obtained from Brenntag StinnesLogistics can be used.

In addition, MN51 can be combined with the polypeptides provided hereinto form a composition that elicits an anti-self response whenadministered to a mammal. MN51 (MONTANIDE® Incomplete SEPPIC Adjuvant(ISA) 51) as well as MN720 are available from Seppic (Paris, France).MN51 contains mannide oleate (MONTANIDE® 80, also known as anhydromannitol octadecanoate) in mineral oil solution (Drakeol 6 VR).MONTANIDE® 80 is a limpid liquid with a maximum acid value of 1, asaponification value of 164-172, a hydroxyl value of 89-100, an iodinevalue of 67-75, a maximum peroxide value of 2, a heavy metal value lessthan 20 ppm, a maximum water content of 0.35%, a maximum color value of9, and a viscosity at 25° C. of about 300 mPas. MONTANIDE® associatedwith oil (e.g., mineral oil, vegetable oil, squalane, squalene, oresters) is known as MONTANIDE® ISA. Drakeol 6 VR is a pharmaceuticalgrade mineral oil. Drakeol 6 VR contains no unsaturated or aromatichydrocarbons, and has an A.P.I. gravity of 36.2-36.8, a specific gravityat 25° C. of 0.834-0.838, a viscosity at 100° F. of 59-61 SSU or10.0-10.6 centistokes, a refractive index at 25° C. of 1.458-1.463, abetter than minimum acid test, is negative for fluorescence at 360 nm,is negative for visible suspended matter, has an ASTM pour test value of0-15° F., has a minimum ASTM flash point of 295° F., and complies withall RN requirements for light mineral oil and ultraviolet absorption.MN51 contains about 8 to 12 percent anhydro mannitol octadecanoate andabout 88 to 92 percent mineral oil. MN51 is a clear yellow liquid havinga maximum acid value of 0.5, a saponification value of 16-20, a hydroxylvalue of 9-13, a maximum peroxide value of 2, an iodine value of 5-9, amaximum water content of 0.5 percent, a refractive index at 25° C.between 1.455 and 1.465, a density at 20° C. of about 0.85, and aviscosity at 20° C. of about 50 mPas. The conductivity of a 50:50mixture of MN51 and saline is less than 10 μScm⁻¹.

Other adjuvants include immuno-stimulating complexes (ISCOMs) that cancontain such components as cholesterol and saponins. ISCOM matrices canbe prepared and conjugated to Cu²⁺ using methods such as those describedherein. Adjuvants such as FCA, FIA, MN51, MN720, and Al(OH)₃ arecommercially available from companies such as Seppic, Difco Laboratories(Detroit, Mich.), and Superfos Biosector A/S (Vedbeak, Demark).

In some embodiments, a composition also can contain one or moreadditional immunostimulatory components. These include, withoutlimitation, muramyldipeptide (e.g.,N-acetylmuramyl-L-alanyl-D-isoglutamine; MDP), monophosphoryl-lipid A(MPL), and formyl-methionine containing tripeptides such asN-formyl-Met-Leu-Phe. Such compounds are commercially available fromSigma Chemical Co. (St. Louis, Mo.) and RIBI ImmunoChem Research, Inc.(Hamilton, Mont.), for example.

The compositions provided herein can contain any ratio of adjuvant topolypeptide. The adjuvant:antigen ratio can be 50:50 (vol:vol), forexample. Alternatively, the adjuvant:antigen ratio can be, withoutlimitation, 90:10, 80:20, 70:30, 64:36, 60:40, 55:45, 40:60, 30:70,20:80, or 90:10.

An effective amount of any composition provided herein can beadministered to a host. The term “effective” as used herein refers toany amount that induces a desired immune response while not inducingsignificant toxicity in the host. Such an amount can be determined byassessing a host's immune response after administration of a knownamount of a particular composition. In addition, the level of toxicity,if any, can be determined by assessing a host's clinical symptoms beforeand after administering a known amount of a particular composition. Itis noted that the effective amount of a particular compositionadministered to a host can be adjusted according to a desired outcome aswell as the host's response and level of toxicity. Significant toxicitycan vary for each particular host and depends on multiple factorsincluding, without limitation, the host's disease state, age, andtolerance to pain.

In addition, any composition described herein can be administered to anypart of the host's body. A composition can be delivered to, withoutlimitation, the joints, nasal mucosa, blood, lungs, intestines, muscletissues, skin, or peritoneal cavity of a mammal. In addition, acomposition can be administered by intravenous, intraperitoneal,intramuscular, subcutaneous, intrathecal, or intradermal injection, byoral or nasal administration, by inhalation, or by gradual perfusionover time. In a further example, an aerosol preparation of a compositioncan be given to a host by inhalation. The duration of treatment with anycomposition provided herein can be any length of time from as short asone day to as long as the life span of the host (e.g., many years). Forexample, a polypeptide can be administered once a month for three monthsor once a year for a period of ten years. It is also noted that thefrequency of treatment can be variable. For example, a polypeptide canbe administered once (or twice, three times, etc.) daily, weekly,monthly, or yearly.

Any method can be used to determine if a particular immune response isinduced. For example, antibody responses against particular antigens canbe determined using an immunological assay (e.g., an ELISA). In such anassay, the wells of a microtiter plate can be coated with C5 andincubated with serum from a mammal treated with a composition designedto produce anti-C5 antibodies in that mammal, and the presence orabsence of anti-C5 antibodies can be determined using a labeled anti-ratIgG. In addition, clinical methods that can assess the degree of aparticular disease state can be used to determine if a desired immuneresponse is induced. For example, a reduction in inflammation canindicate a desired immune response in a patient treated with acomposition designed to produce anti-C5 antibodies. To support anindication of a desired immune response, anti-C5 antibody levels in ablood sample from such a patient can be measured using the ELISAtechnique described above.

Articles of Manufacture

The invention also provides articles of manufacture that can includepolypeptides and compositions provided herein. Components and methodsfor producing articles of manufacture are well known. An article ofmanufacture can include, for example, one or more polypeptides thatinduce production of an anti-self C5 antibody (e.g., one or morepolypeptides containing a self C5 amino acid segment and a non-selfamino acid segment such as a non-self C5 amino acid segment). Inaddition, an article of manufacture further may include, for example,buffers or other control reagents for treating or monitoring aninflammatory condition. In some embodiments, such articles ofmanufacture can include a label or instructions indicating that thepolypeptides contained therein are effective for treating aninflammatory condition.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1 Production and Purification of a Mouse C5aPolypeptide

A mouse pro-C5 DNA sequence (SEQ ID NO:1) is shown in FIG. 1, and theamino acid sequence of mouse pro-C5 including the signal peptide (SEQ IDNO:2) is shown in FIG. 2. The coding region for mouse C5a was isolatedby PCR amplification from a total mouse liver cDNA library. The PCRfragment was ligated into the bacterial expression vector pMAL-p2x (FIG.3; New England Biolabs, Beverly, Mass.), adjacent to a sequence encodinga portion of maltose binding protein (MBP). The coding region for theMBP-C5a fusion polypeptide was then amplified from this vector by PCR.To facilitate eventual purification of the fusion polypeptide, the 5′PCR primer also encoded six histidine residues. In addition, the 5′primer contained an EcoRI site, and the 3′ primer contained a SalI sitefor cloning purposes. The PCR product (sequence shown in FIG. 4) wastransferred into a mammalian expression vector, the episomallymaintained pCEP-Pu2, which contains a signal sequence from aimmunoglobulin variable region. The SalI site of the PCR product wasligated into the Yh6J site of the pCEP-Pu2 vector, resulting indestruction of both sites. The nucleotide sequence of the PCR productencoding the fusion polypeptide is set forth in SEQ ID NO:3, and theamino acid sequence of the fusion polypeptide is set forth in SEQ IDNO:4.

Example 2 Expression and Purification of MBP-C5a from Mammalian Cells

The pCEP-Pu2 construct containing the 6His-MBP-C5a fusion polypeptidewas transfected into 293-EBNA cells for over-expression and purificationof the secreted fusion polypeptide. Mammalian EBNA-293 cells weretransfected by lipofection and cultured in Dulbecco's Modified EagleMedium (DMEM) supplemented with 5% fetal calf serum, 0.5 μg/mlpuromycin, 50 μg/ml gentamicin, and 100 μg/ml geneticin. Growing cellswere expanded, and conditioned media were collected every 4-5 days.Cells and debris were removed by centrifugation.

The fusion polypeptide contained six histidine residues to enablepurification using Ni-NTA agarose. A 0.75 ml aliquot of Ni-NTA agaroseslurry (QIAGEN GmbH, Germany) containing 0.35 ml beads in 20% ethanolwas added per 500 ml of conditioned media. After overnight incubation at4° C. on a shaker, the Ni-NTA agarose was pelleted by centrifugation for10 minutes at about 2000 g and transferred to columns. The beads werewashed with PBS, pH 7.4, in the presence of 1 M NaCl and 0.1% Tween-20,and eluted with 100 mM imidazole (Merck, Germany) in 20 mM Tris, pH 8.0,with 0.1 M NaCl, according to the manufacturer's instructions.Polypeptide-containing fractions were pooled and dialyzed against PBS,pH 7.4, using a membrane with a molecular weight cut-off of12,000-14,000 Da (Spectra/Por Membranes, Spectrum Laboratories, Inc.,Rancho Dominguez, Calif.). If necessary, samples were concentrated usinga Macrosep 10K centrifugal device (PALL Gelman Laboratory). The finalpolypeptide concentration was estimated with Bradford assay (BIO-RADProtein Assay, Bio-Rad Laboratories, Hercules, Calif.). The cellsproduced the 6His-MBP-C5a fusion polypeptide at a level of about 2mg/liter. A Rainbow protein molecular weight marker (AmershamInternational, Buckinghamshire, England) was used for size estimation.The polypeptide migrated at the expected size of 53 kD. The amino acidsequence of the fusion polypeptide is shown in FIG. 5.

Example 3 Effect of MBP-C5a on Murine Collagen-Induced Arthritis

Purified 6His-MBP-C5a polypeptide in PBS was combined with CompleteFreund's Adjuvant (CFA) or Incomplete Freund's Adjuvant (IFA) in a 1:1ratio immediately before use. The solution was repeatedly drawn up anddown in a Hamilton syringe (Hamilton Corp., Reno, Nebr.) equipped withan 18 G needle, and was mixed until a white emulsion with rheologicalcharacteristics similar to an ointment was formed. A 23 G needle wasplaced on the syringe for administration of the mixture. A controlmixture containing equal volumes of PBS and CFA or IFA also wasprepared.

Twelve week-old male QB (Balb/c X B10.Q) F1 mice were divided into twogroups of 14 or 16 animals each, and were subcutaneously injectedbetween the scapulae on day −21 with either 100 μg of MBP-C5a emulsifiedin CFA or with the control mixture. Blood samples also were taken on day−21, and the sera were stored at −20° C. Animals received boosterinjections of either 50 μg of MBP-C5a in IFA or the control mixture onday −3 and on day 28. Injections typically were administered in a volumeof 100 to 200 μL.

To induce arthritis, mice were intradermally injected at the root of thetail on day 0 with 100 μg of pepsin-digested CII emulsified 1:1 in CFA.Arthritis was expected to develop between days 28 and 56. Animalsreceived a booster injection of CII emulsified in IFA on day 35, andblood samples were taken on day 35. Animals were examined at least threetimes weekly from day 14 until day 90, when the experiment was ended.Animals were scored blindly using a scoring system based on the numberof inflamed joints in each paw. Inflammation was defined by swelling andredness. In this scoring system, each inflamed toe or knuckle was givenone point, whereas an inflamed wrist or ankle was given five points.This resulted in a score of 0-15 for each paw (5 toes+5 knuckles+1wrist/ankle), and 0-60 for each mouse. The experiment was terminated onday 90 or when it was deemed appropriate based on development ofarthritis. No mice exhibited signs of abnormal fur status, stereotypicor behavioral changes, infection, or other severe or unexpected sideeffects apart from symptoms normally occurring in connection with theexpected arthritis disease. Animals were anesthetized with enfluran(Forene®)/oxygen, and blood was obtained by reorbital puncture. Serumwas collected and stored at −20° C. Sera collected on days −21, 35, andat the end of the study were evaluated for anti-CII and anti-C5aantibody levels using an ELISA method.

The Mann-Whitney test was used to analyze the scoring data, and areasunder the curve and chi-square values were used to analyze thesignificance of the incidence of arthritis. On day 28 after the firstcollagen treatment, seven of the 14 mice in the control group (50%)displayed inflammation, while only two of the 16 mice (12.5%)pre-treated with MBP-C5a displayed inflammation (FIG. 6). These dataresulted in a chi-square P value of 0.025. The cumulative incidence ofinflammation (as of day 67) was 14 out of 14 in the control group and 11out of 16 in the group pretreated with MBP-C5a (P=0.022). Thus, theincidence of collagen-induced arthritis was significantly differentbetween the two groups.

A mean arthritis score based on the amount of inflammation was plottedfor each group between days 1 and 90 (FIG. 7). The maximum one-mousescore for each group was 60. At the end of the study, the maximumseverity resulted in a mean value of 38.6 in the control group and 19.0in the group pretreated with MBP-C5a (P=0.0085; FIG. 8). The area underthe curve resulted in a mean value of 485.9 in the control group and168.9 in the group pretreated with MBP-C5a (P=0.0012; FIG. 9). Theseresults demonstrated that treatment with the MBP-C5a fusion polypeptideresulted in decreases in the incidence and severity of collagen-inducedarthritis in these animals.

Example 4 Effect of MBP-C5a on Chronic Collagen-Induced Arthritis inQB-BC Mice

Purified MBP-C5a polypeptide in PBS was combined with CFA or IFA asdescribed above. A control mixture containing equal volumes of PBS andCFA or IFA also was prepared.

Eight- to ten-week-old male and female QB-BC (B10.Q(Balb/c X B10.Q))mice were intradermally injected at the root of the tail with 100 μg ofpepsin-digested CII emulsified 1:1 in IFA. After 35 days, mice receiveda second injection with 50 μg of pepsin-digested CII in IFA. Mice werethen scored at least 3 times a week for development of chronicarthritis. Many, but not all of the mice developed a chronic relapsingdisease course characterized by periods of recurrence of activearthritis. These relapses appeared without prediction, lasted for a fewweeks at a time, and seemed to occur on a life-long basis. Thevariability of the arthritis effect in the cohort was mainly due togenetic heterogeneity (the mice were N2 animals, due to an experimentaldesign aimed at mimicking the genetic situation in humans).

To coordinate the recurrence of relapses, mice were reimmunized with CIIduring the chronic relapsing phase. When the mice were 12-14 months old,animals that had chronic relapsing disease but that presently had noactive disease relapse were randomly mixed and sorted into fourequal-sized groups. To induce a controlled arthritis relapse, the micewere subcutaneously injected between the scapulae on day −21 (day 259after the first CII immunization) with either 100 μg of MBP-C5a in PBS(n=24) or with PBS only, each emulsified 1:1 in CFA (n=12). Animalsreceived a booster injection of either 50 g of MBP-C5a in PBS or PBSalone (each in CFA) on day −3. On day 0 (day 280 after the first CIIimmunization), animals in both groups were intradermally injected at theroot of the tail with 50 μg of pepsin-digested CII emulsified 1:1 inIFA. A second booster injection of 50 μg MBP-C5a in PBS, or PBS only,was administered on day 21 (day 301 after the first CII immunization).In addition, blood samples were obtained by reorbital puncture on day−21 and day 0.

Animals were scored blindly using the scoring system described above,resulting in an arthritis score of 0-60 for each mouse. The experimentwas terminated at day 358 after the first CII immunization. Thesestudies revealed that while all of the control mice exhibited arthritissymptoms, especially around day 40, only 50% of the mice immunized withMBP-C5a exhibited chronic arthritis (p<0.05; FIG. 10). In addition, themean arthritis score was determined for the two groups from thebeginning of the study (i.e., from the first injection of CII) throughthe end of the study more then 358 days later and day 78 afterreinduction or relapse (FIG. 11). The mean arthritis score was notsignificantly different between the two groups until immunization withMBP-C5a, after which the mean arthritis score for the treated group wassignificantly lower than for the control group (P<0.05; FIG. 11). Theseresults demonstrate that the MBP-C5a fusion polypeptide was able toreduce the incidence of chronic relapsing arthritis in the treatedanimals.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. A composition comprising a polypeptide, wherein said polypeptidecomprises a self C5 amino acid segment and a non-self amino acidsegment, wherein the length of said non-self amino acid segment is lessthan 350 amino acids. 2-15. (canceled)